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Am J Physiol Heart Circ Physiol 289: H1047-H1053, 2005. First published April 15, 2005; doi:10.1152/ajpheart.01071.2004
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Tempol therapy attenuates medial smooth muscle cell apoptosis and neointima formation after balloon catheter injury in carotid artery of diabetic rats

D. K. Jagadeesha,1,2 Timothy E. Lindley,1 Jason DeLeon,1 Ram V. Sharma,1,2 Francis Miller,2,3,4 and Ramesh C. Bhalla1,2

1Department of Anatomy and Cell Biology, 2The Cardiovascular Center, 3Department of Internal Medicine, and 4Free Radical and Radiation Biology Program, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, Iowa

Submitted 19 October 2004 ; accepted in final form 12 April 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Accumulating data support the hypothesis that reactive oxygen species (ROS) play a critical role in the vascular complications observed in diabetes. However, the mechanisms of ROS-mediated vascular complications in diabetes are not clear. We tested the hypothesis that ROS-mediated increase in proapoptotic factor Bax expression leads to medial smooth muscle cell (SMC) apoptosis that is associated with neointima formation. We used a fructose-rich diet for 4 wk to model Type 2 diabetes in rats. SOD mimetic membrane-permeable 4-hydroxy-2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempol, 1 mM) was administered in drinking water to scavenge superoxide starting 1 day before surgery and continued during the duration of the experiment. Vascular injury resulted in a significant increase in medial SMC apoptosis that was associated with neointima formation. The number of medial SMC positive for Bax immunostaining significantly increased in injured arteries compared with uninjured arteries. Superoxide scavenging by Tempol treatment inhibited both the Bax-positive index as well as the apoptotic index of medial SMC in response to vascular injury. Tempol treatment inhibited apoptotic loss of medial SMC, thus increasing their density in the injured arteries. These alterations in the media were associated with a marked decrease in neointima formation in injured arteries. We conclude that Bax expression may play an important role in vascular SMC apoptosis and, finally, that this regulatory mechanism is redox sensitive.

vascular smooth muscle; vascular injury; Type 2 diabetes; vascular smooth muscle apoptosis; superoxides; Bax protein

TYPE 2 DIABETES is the most critical risk factor for all patients with coronary artery disease. The pathophysiology is associated with increased vascular proliferative response in diabetic patients and is characterized by late morbidity and mortality (21). Functional derangement of vascular smooth muscle cells (SMCs) as a manifestation of cardiovascular complications of diabetes has been observed (1, 32, 38). During the past few years, convincing evidence has emerged indicating that increased oxidative stress plays a significant role in the pathogenesis of vascular complications associated with type 2 diabetes (37). Indeed, Type 2 diabetes is characterized by increased oxidative stress (39). It is postulated that glucose is prone to oxidation to generate reactive oxygen species (ROS), which may cause increased peroxidation (43).

The hyperglycemia and increased fatty acids in the bloodstream observed in Type 2 diabetes can cause oxidative stress and damage to the mitochondrial membrane (7). Systemic antioxidant therapy with probucol has been shown to reduce intima formation and restenosis after vascular injury (27). However, a limitation of most of the antioxidants used so far, including probucol, has been the need to administer the drug for 2 to 4 wk before angioplasty to demonstrate its beneficial effect in reducing neointima formation (9, 27). Therefore, we examined the effects of the SOD mimetic membrane-permeable 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol, Sigma) administered 1 day before surgery and continued during the entire experimental period. This low-molecular-weight cyclic nitroxide acts in a catalytic fashion and is regenerated in the process of quenching superoxide. It has been shown to reduce oxidative stress-mediated injury during ischemia-reperfusion (10) and inflammation (18).

Emerging evidence suggests that increased ROS production leads to vascular SMC apoptosis following injury (24). Apoptosis is a highly regulated cell deletion process characterized by cellular and nuclear fragmentation. SMC apoptosis associated with significant intimal hyperplasia has been observed in several models of vascular injury (13, 15, 25, 31, 36). In animal models of balloon vascular injury, marked superoxide production and massive apoptosis of medial SMCs were observed within the first hour, resulting in loss of cell density in the medial layer (30). Apoptosis of medial SMCs precedes neointima formation, suggesting that cell death of medial SMCs may trigger cell migration and proliferation to form neointima (25).

Although signal transduction pathways involved in redox-linked vascular SMC apoptosis are poorly defined, recent evidence suggests that Bcl family genes may play a crucial regulatory role. Proapoptotic Bcl family members such as Bax and Bak cause induction of apoptosis, disrupting outer mitochondrial membrane integrity (19, 42). On the other hand, antiapoptotic members such as Bcl-2 and Bcl-xL prevent apoptosis by heterodimerization with Bax and Bak, thus preventing outer mitochondrial membrane disruption (3, 44). Given the link between increased ROS and medial SMC apoptosis as well as emerging evidence that Bax may play a role in apoptosis, we hypothesized that antioxidant therapy with Tempol would inhibit medial SMC apoptosis and neointima formation in balloon-injured rat carotid arteries by inhibiting Bax induction. To test this hypothesis, we examined the effects of antioxidant Tempol in balloon injury-induced expression of Bax on medial SMC apoptosis and neointima formation in rats. These findings provide further evidence for the role of ROS in the pathogenesis of neointima formation after vascular injury. These data suggest that inhibition of redox-sensitive signaling pathways by Tempol may represent an important therapeutic approach in the treatment of vascular proliferative diseases in diabetes.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. All experiments were performed within the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the Animal Care and Use Committee at The University of Iowa. Adult male Sprague-Dawley rats weighing 300–325 g were used for this study. Animals were housed in a room with a 12:12-h light-dark cycle and an ambient temperature of 22°C. Animals were fed a fructose-rich diet (60%; Harlan) for 30 days before the surgery to induce type 2 diabetes as has been described earlier (14, 16).

Plasma blood glucose and insulin estimation. Blood plasma was collected from nonfasting animals by eye puncture to measure plasma insulin using the ultrasensitive rat insulin ELISA kit (Crystal Chem). Blood glucose was measured with a standardized portable glucometer (ACCU-Chek; Roche Diagnostics).

Surgical procedure and drug treatment protocol. Catheter balloon injury of the left common carotid artery was performed essentially as described previously (8). In brief, male Sprague-Dawley rats weighing 300–325 g were preanesthetized with halothane (Halocarbon, River Edge, NJ) and then anesthetized by intraperitoneal injection of ketamine (10 mg/kg). To produce balloon injury, we exposed the bifurcation of the left common carotid artery through a midline incision and temporarily ligated the left common and internal carotid arteries. A 2-F embolectomy balloon catheter (Baxter Edwards Lifesciences, Irvine, CA) was introduced into the isolated common carotid artery through an arteriotomy site in the external carotid artery and advanced to the distal ligation of the common carotid artery. The balloon was inflated with air and drawn toward the arteriotomy three times to produce a distending, deendothelializing injury. After the catheter was withdrawn, the proximal end of the external carotid artery was ligated, and blood flow was restored. The surgical incision was closed, and the rats were allowed to recover from anesthesia. The right uninjured artery was used as control tissue. Although investigators in our laboratory (8) and others (5, 23) have used uninjured contralateral artery as a control, it should be kept in mind that a sham procedure in which the control artery is stretched and handled without injury to the endothelium may represent a better control tissue. Animals were allowed free access to water, and a fructose-rich diet was given to the experimental group throughout the duration of the experiment. For one group of animals, Tempol (1 mM), freshly dissolved in drinking water, was administered orally. Tempol administration was initiated 1 day before surgery and was continued throughout the experiment.

Tissue preparation. Fourteen days after angioplasty, animals were anesthetized as described in Surgical procedure and drug treatment protocol. The neck and thorax were opened to expose the carotid arteries, the heart, and the great vessels. Carotid arteries were fixed by perfusion of 200 ml of saline followed by 500 ml of saline containing 4% paraformaldehyde through a large cannula placed in the left ventricle over a 20-min period. After perfusion fixation, the right and left carotid arteries were excised, excess periadventitial tissue was trimmed, and arteries were placed in 4% paraformaldehyde for 48 h before paraffin embedding.

Histomorphometric measurement. For morphometric analysis, paraffin-embedded sections were used for each of the arterial specimens. Cross sections (6 µm) were obtained from uninjured and injured carotid arteries from control and Tempol-treated groups and stained with hematoxylin and eosin. The images were digitized using an Olympus BX 51 microscope (Leeds Precision Instruments), and photographs were captured using a digital camera. Morphometric analysis of these arterial sections was performed as described previously (8). For each arterial cross section, the intimal and medial areas were measured and the ratio of intima to media area was calculated. Morphometric analysis was performed using NIH Image analysis software.

In situ labeling of fragmented DNA. Formalin-fixed and paraffin-embedded tissue sections were deparaffinized, rehydrated, and treated with proteinase K (20 µg/ml), and endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol. In situ end labeling of apoptotic cells was performed as described in the protocol provided by the manufacturers of the in situ apoptosis detection kit (Trevigen). In brief, after equilibration, end labeling with digoxigenin-dUTP by TdT enzyme in buffer was carried out for 1 h at 37°C in a humidifying chamber. After treatment with stop/wash buffer, sections were incubated with anti-digoxigenin antibody-peroxidase conjugate, rinsed, and stained with diaminobenzidine tetrahydrochloride. Negative controls were incubated with PBS instead of TdT enzyme, and positive controls were treated with DNase 1. Sections were counterstained with 1% methyl green. Apoptotic cells were counted in the medial layer of a cross section of the artery, and apoptotic index was calculated as the number of labeled nuclei per the number of total nuclei x 100. After DNA nick-end labeling (TUNEL), tissue sections in which apoptosis was detected in the SMCs were identified by immunostaining with an alkaline phosphatase-conjugated monoclonal antibody to {alpha}-smooth muscle actin.

Immunohistochemistry for activated caspase-3 and Bax. To compare and extend the responses estimated by the TUNEL assay, we employed caspase-3 immunohistochemistry for the detection of apoptosis of vascular SMCs by using antibodies that specifically recognize only the large subunit of activated caspase-3, as described previously (5, 11). Tissue sections (6 µm) were mounted on poly-L-lysine-coated glass slides. After deparaffinization and rehydration, sections were blocked for endogenous peroxidase activity with 3% hydrogen peroxidase in methanol. Sections were then incubated in an anti-caspase-3 primary antibody that specifically recognizes the large fragment (17 kDa) of activated caspase-3 (1:50; Calbiochem, San Diego, CA) and a rabbit polyclonal anti-mouse Bax-{alpha} antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) separately overnight at 4°C. After being washed, the slides were incubated with biotinylated secondary antibody for 30 min at room temperature. After two washes in PBS, sections were incubated in streptavidin-horseradish peroxidase (Vector Laboratories), washed two more times in PBS, and then incubated for 5 min in the dark with diaminobenzidine. Sections were counterstained using 1% methyl green. Data are expressed as the number of Bax-positive and activated caspase-3-positive cells per the total number of nuclei x 100.

Estimation of SMC density in media. For the assessment of cellularity, paraffin-embedded arterial sections were stained with Hoechst 33258 (Molecular Probes) for 2 h at 37°C. Images were captured with a x40 objective on an epifluorescent microscope in three to four different fields, and the area was calculated using a computerized program. The cellularity in the medial layer was expressed as the Hoechst index, defined as the positive nuclear staining averaged for one fixed area (0.1 mm2) in the medial layer. Histological samples were randomly examined between treatment groups by an independent investigator who was blind to the treatment protocol.

Superoxide measurement by enhanced chemiluminescence. The carotid arteries of rats fed a normal diet were immediately frozen in OCT on dry ice. Frozen sections (40 µm) were incubated in 1 ml of PBS containing 5 µM lucigenin (bis-N-methylacridinium nitrate) for 2 min in the dark. The sample was then placed in a luminometer (Monolight 2010) for measurement of relative light units emitted every 30 s for 4 min. The values obtained between 2 and 4 min were averaged to determine basal emission levels. NADPH (50 µM; Sigma) was then added, and the relative light units emitted were again recorded every 30 s for 4 min. The values obtained between 2 and 4 min were averaged, and the basal emission level was subtracted.

Statistical analysis. Data are expressed as means ± SE or means (SD), as indicated. The statistical comparison was performed using one-way analysis of variance (ANOVA) followed by the Bonferroni test. A value of P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Fructose-rich diet increases plasma glucose and insulin levels. The effects of fructose-rich diet on plasma glucose and insulin concentration are shown in Fig. 1. The glucose levels were significantly higher in animals fed fructose-rich diet compared with animals fed normal chow [fructose, 177 (50) vs. normal, 136 (16) mg/dl, mean (SD); P < 0.05, n = 18]. Although the insulin levels were higher in animals fed fructose-rich diet compared with animals fed normal chow diet, they did not reach statistical significance [fructose, 4.8 (1.4) vs. normal, 3.9 (1.8) ng/dl, mean (SD); n = 15]. It is possible that the variability in glucose and insulin levels was increased because animals were not fasted before blood samples were collected.



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  		Fig. 1. Analysis of plasma glucose and insulin levels in rats fed high-fructose diet and rats fed normal chow. Rats were fed high-fructose diet for 30 days before surgery. Blood was collected by ocular puncture at the time of carotid artery balloon catheter injury. A: blood glucose was estimated with a standardized portable glucometer. Values are means (SD); n = 18. *P < 0.05, high-fructose diet vs. normal chow. B: plasma insulin levels were measured using an ultrasensitive rat insulin ELISA kit. Values are means (SD); n = 15. Three of the samples were lost, and insulin levels could be estimated for 15 samples.

 
Tempol inhibits vascular injury-induced increases in apoptosis of medial SMC. Balloon distension and endothelium denudation injury resulted in an increase in medial SMC apoptosis characterized by shrinkage and nuclear condensation visualized by the TUNEL assay that stains nuclei containing nicked DNA, a characteristic signifying the early stages of apoptotic cell death. Quantification of the apoptotic SMCs in the medial layer showed a significant increase in apoptotic index in the injured artery compared with the uninjured artery of the diabetic rats (injured, 12.9 ± 0.44 vs. uninjured, 2.5 ± 0.18%; P < 0.05, n = 5) (Fig. 2A). Tempol treatment significantly inhibited apoptosis of the medial SMCs of injured arteries (injured, 12.9 ± 0.4 vs. injured + Tempol, 5.2 ± 0.3%; P < 0.05, n = 5) (Fig. 2A).



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  		Fig. 2. Quantification of apoptosis in the medial layer of the carotid arterial sections. A: apoptotic cells were detected using Apop-Tag, an in situ apoptosis detection kit. The fraction of positive nuclei was calculated as (number of TUNEL-positive cells/total number of cells) x 100. Tempol treatment inhibited apoptosis of medial smooth muscle cells (SMCs) in injured arteries. Results are expressed as means ± SE; n = 5. *P < 0.05, injured vs. uninjured arteries. #P < 0.05, injured vs. Tempol-treated injured arteries. {dagger}P < 0.05, uninjured vs. Tempol-treated injured arteries. Uninj, uninjured arteries; inj, untreated injured arteries; inj+temp, Tempol-treated injured arteries. B: paraffin sections were immunostained with an anti-caspase-3 antibody that specifically detects the large fragment (17 kDa) of activated caspase-3. Data are expressed as (number of activated caspase-3-positive cells/total number of nuclei) x 100. Values are means ± SE; n = 5. *P < 0.05, injured vs. uninjured arteries. {dagger}P < 0.05, injured vs. Tempol-treated injured arteries.

 
In the initial experiments, we observed that Tempol treatment did not effect the TUNEL-positive medial SMC indexes in uninjured arteries (data not shown). In addition to TUNEL assay, we employed immunohistochemistry to detect apoptosis of SMCs by using antibodies that specifically recognize the large subunit of activated caspase-3. Activated caspase-3-positive apoptotic cells were quantified, and apoptotic indexes (apoptotic cells/total cells x 100) were calculated. The activated caspase-3-positive apoptotic index was significantly increased in the injured arteries compared with the uninjured control arteries (uninjured, 3.13 ± 0.42 vs. injured, 18.8 ± 0.45%; P < 0.05, n = 5) (Fig. 2B). Tempol treatment significantly reduced the apoptotic indexes in the injured arteries (injured, 18.8 ± 0.45 vs. injured + Tempol, 7.51 ± 0.62%; P < 0.05, n = 5). These data demonstrate that inhibition of oxidative stress by Tempol treatment attenuated medial SMC apoptosis and suggest that signaling pathways associated with ROS are involved in medial SMC apoptosis induced by vascular injury.

Tempol inhibits loss of cellularity after vascular angioplasty. Next, we examined whether inhibition of medial SMC apoptosis increases medial SMC density. Compared with that in vessels without mechanical injury, cell density in the medial layer was significantly decreased after injury (uninjured, 23.0 ± 2.7 vs. injured, 10.9 ± 1.5 SMCs/0.1 mm2; P < 0.05, n = 4), and Tempol treatment attenuated the injury-induced decrease in medial SMC density (injured, 10.9 ± 1.5 vs. injured + Tempol, 18.0 ± 3.0 SMCs/0.1 mm2; P < 0.05, n = 4) (Fig. 3).



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  		Fig. 3. Medial SMC density. Rat carotid arterial sections of all treatment groups were viewed en face after staining with Hoechst 33258. Images were captured with a x40 objective on an epifluorescent microscope. The cellularity in the media is expressed as Hoechst index, defined as the positive nuclear staining averaged for a fixed area. Values are means ± SE; n = 5. *P < 0.05, injured vs. uninjured arteries. {dagger}P < 0.05, injured vs. Tempol-treated injured arteries.

 
Bax expression is inhibited by Tempol treatment. The Bax protein is expressed in medial SMCs at low levels. Therefore, to elucidate the mechanisms that may lead to apoptosis of medial SMCs after injury of the carotid artery, we examined the expression of Bax in medial SMCs. The proportion of medial SMCs positive for Bax immunostaining significantly increased in the injured arteries compared with the uninjured arteries of diabetic rats (injured, 6.0 ± 0.56 vs. uninjured, 2.6 ± 0.4%; P < 0.05, n = 5) (Fig. 4). The proportion of Bax-positive cells in the media was significantly decreased by Tempol treatment (injured, 6.0 ± 0.56 vs. injured + Tempol, 4.0 ± 0.53%; P < 0.05, n = 5) (Fig. 4) and was not significantly different from that in uninjured vessels. These results demonstrate that oxidant stress-mediated Bax expression plays a critical role in the induction of medial SMC apoptosis and that antioxidant therapy can inhibit Bax expression and vascular SMC apoptosis in response to vascular injury.



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  		Fig. 4. Effect of Tempol on Bax immunostaining index in medial SMCs. Immunohistochemical staining of Bax protein of rat carotid arterial sections was performed in all treatment groups. Injured artery exhibits an increase in Bax immunoreactivity compared with the control and Tempol treatment, which significantly inhibited Bax expression in medial cells. Data are expressed as (number of Bax-positive cells/total number of cells) x 100. Values are means ± SE; n = 5. *P < 0.05, injured vs. uninjured arteries. {dagger}P < 0.05, injured vs. Tempol-treated injured arteries.

 
Antioxidant Tempol inhibits ROS generation in injured arteries. To verify the effectiveness of oral administration of Tempol to attenuate the elevation in superoxide levels in the injured vessel, we estimated superoxide levels by using the lucigenin-enhanced chemiluminescence method. Injury produced a significant increase in ROS levels in carotid arteries, and Tempol treatment prevented this increase (Fig. 5). These data show an increase in superoxide levels following vascular injury, and Tempol treatment can effectively inhibit superoxide levels.



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  		Fig. 5. Effect of Tempol on superoxide production in balloon-injured arteries. Frozen carotid arterial sections (40 µm) of uninjured, injured, and Tempol-treated arteries were incubated in 1 ml of PBS containing 5 µM lucigenin. Relative light units (RLU) obtained between 2 and 4 min were averaged, and the basal emission was subtracted. Values are means ± SE; n = 5. *P < 0.05, injured vs. uninjured arteries. {dagger}P < 0.05, injured vs. Tempol-treated injured arteries. #P < 0.05, uninjured vs. inj+temp.

 
Tempol treatment attenuates neointima formation in balloon-injured rat carotid artery. Two weeks after injury, a concentric neointimal lesion was found at the site of injury (Fig. 6). The neointima consisted mainly of SMCs as determined by immunostaining for {alpha}-SMC actin. Quantitative analysis of the intima-to-media ratio of pressure-fixed vessel cross sections showed that the ratio was significantly increased (P < 0.05) in injured compared with uninjured arteries (uninjured, 0.12 ± 0.008 vs. injured, 0.73 ± 0.04), and Tempol treatment significantly reduced the neointima-to-media ratio in injured arteries of diabetic rats (injured, 0.73 ± 0.04 vs. injured + Tempol, 0.22 ± 0.04; P < 0.05, n = 5).



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  		Fig. 6. Photomicrographs showing intimal lesions 14 days after balloon injury. Representative histological sections of pressure-perfused carotid artery were taken at 14 days after balloon catheter injury and stained with hematoxylin and eosin. Representative photographs show uninjured (A), injured (B), and Tempol-treated injured arteries of rats (C). Injury resulted in development of neointima formation, and Tempol treatment inhibited neointima formation.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The possible targets of ROS in SMCs and their relation to neointima formation in diabetes mellitus were discussed in a recent review (37). However, the mechanism by which ROS controls neointima formation remains unclear. Our studies demonstrate that the membrane-permeable SOD mimetic Tempol is an effective pharmacological compound in inhibiting injury-induced superoxide levels, SMC apoptosis, and neointima formation at the site of injury. We propose that after vascular injury, ROS play a central role in the vascular remodeling process, and we further speculate that antioxidant therapy may retard injury-induced remodeling of the vessel wall in diabetes.

Vascular SMC apoptosis plays a critical role in the pathogenesis of a number of cardiovascular diseases (40). The occurrence of SMC apoptosis after balloon angioplasty has been demonstrated in both animal and human models (4, 6, 13, 15, 20, 25). Previous studies with the balloon catheter injury of rat carotid artery have demonstrated extensive apoptosis of medial SMCs, and subsequent neointima formation reaches maximum by day 14 postsurgery (30). Therefore, we have examined in this study the effects of vascular injury at 14 days postinjury, by which time neointima formation is well developed. It has been postulated that loss of cellularity due to apoptosis of medial SMCs may play a critical role in triggering a signaling pathway that would allow the remaining SMCs to reenter the cell cycle for the repopulation of media and subsequent intima formation due to migration and proliferation of medial SMCs (31, 41). However, the mechanisms underlying vascular SMC apoptosis in response to vascular injury are not completely elucidated.

To begin to test the hypothesis that increased ROS play a role in medial SMC apoptosis, we used antioxidant Tempol and quantified apoptotic cells in the medial layer by TUNEL assay and by immunohistochemistry for activated caspase-3 (Fig. 2). We found that 2 wk after vascular injury, there was a significant increase in the apoptotic indexes of medial SMCs in the injured arteries compared with the uninjured arteries. Tempol treatment significantly inhibited (P < 0.05) apoptosis of medial SMCs in the injured arteries. These findings are consistent with a number of reports showing that medial SMC apoptosis plays a significant role in intimal hyperplasia in several models of vascular injury and in atherosclerotic lesions (13, 15, 25, 31, 36). Although a relationship between medial SMC apoptosis and neointima formation has been postulated, a link between medial SMC apoptosis and intimal hyperplasia has been lacking. Next, we examined whether inhibition of medial SMC apoptosis would increase medial SMC density. Our results demonstrate that Tempol treatment prevents the loss of medial SMC density (Fig. 3), which is associated with the inhibition of neointima formation (Figs. 6 and 7). How Tempol increases medial SMC density is not clear at this time. It is possible that Tempol may inhibit the initial loss of medial SMC in response to injury. Alternatively, Tempol may regulate proliferation of the remaining medial SMCs. Further studies are needed to examine the effect of Tempol on the dynamics of medial SMC apoptosis and proliferation at different time intervals immediately following injury. Although there was an association between the extent of medial SMC apoptosis and the extent of neointima formation, we have not demonstrated a cause-and-effect relationship between these two events in this study. It is likely that Tempol may be inhibiting neointima formation by mechanisms independent of those inhibiting SMC apoptosis. ROS has been shown to increase SMC growth and migration (11, 26, 35). It is likely that Tempol may be inhibiting neointima formation by inhibiting SMC migration and proliferation. Future studies using alternate methods to inhibit medial SMC apoptosis and testing its effects on neointima formation are required to further elucidate the role of medial SMC apoptosis in neointima formation.



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  		Fig. 7. Effect of Tempol treatment on intimal hyperplasia. Morphometric analysis was performed using the NIH Image analysis program. Tempol treatment significantly inhibited the intima-to-media ratio. Values are means ± SE; n = 5. *P < 0.05, injured vs. uninjured arteries. {dagger}P < 0.05, injured vs. Tempol-treated injured arteries.

 
The loss of medial SMCs due to apoptosis coincided with intimal hyperplasia. Thus the intima-to-media ratio in the injured rats was 0.73 ± 0.04 and was reduced to 0.22 ± 0.04 in Tempol-treated animals. Morphometric examination 2 wk after vascular injury showed that inhibition of medial SMC apoptosis by Tempol treatment of diabetic rats correlated with reduced neointima formation compared with untreated rats (Fig. 7). These data confirm the previous observations showing that antioxidants inhibit neointima formation after injury (31). Furthermore, our data suggest that the medial SMC apoptotic index and a decreased medial SMC density both have a bearing on the extent of neointima formation. We hypothesize that apoptotic cells send a message to healthy cells to differentiate, leading to cell migration and proliferation. Thus antioxidant Tempol therapy, by maintaining medial SMC density after vascular injury, may minimize the stimulus for healthy cells to migrate and proliferate. Further studies are needed to determine the long-term beneficial effects of antioxidant treatment on intimal hyperplasia.

Previous studies in other tissues have indicated that apoptosis is regulated by a balance between the expression of proapoptotic mediators such as Bax and the antiapoptotic mediators Bcl-x and Bcl-2 (34). However, studies in vascular lesions have demonstrated that Bcl-x is expressed primarily in neointima cells, and the downregulation of Bcl-x by antisense oligonucleotides does not affect the apoptosis of the medial SMCs or their cellularity in injured arteries (33). To elucidate potential mechanisms of ROS-mediated apoptosis, we studied expression of Bax, a proapoptotic protein, in medial SMCs by using immunohistochemistry. The proportion of Bax-positive SMCs in the medial layer was markedly increased at 2 wk postinjury, concomitant with an increase in medial SMC apoptosis, suggesting that modulation of this protein may play a crucial role in apoptotic response to injury. These findings are in agreement with the role of Bax in the initial phase of apoptosis (22) and are consistent with the observations that adenovirus-mediated overexpression of Bax induces apoptosis in many cells (28, 29).

We have observed that the proportion of Bax-positive cells in the medial layer is attenuated by Tempol treatment, with a concomitant inhibition of apoptotic index. These findings suggest that increased ROS generation mediates its effects, at least in part, by increasing Bax expression, resulting in medial SMC apoptosis. Our findings are consistent with previous studies showing that oxidative stress causes apoptosis in vascular SMCs due to an increase in the ratio of Bax to Bcl-2 in the blood vessel wall as estimated using biochemical procedures (2). However, the immunohistochemical techniques used in our study permitted us to estimate the number of Bax-positive cells in the media directly. A number of biochemical functions have been defined for Bax that support its suggested role as a proapoptotic protein, including induction of cytochrome c release from mitochondria (17). The present study has provided evidence that the inhibition of Bax by antioxidant therapy can inhibit medial SMC apoptosis, resulting in the inhibition of intima formation.

Investigators in our laboratory (12) have previously demonstrated that matrix metalloproteinase (MMP)-9 expression is regulated by the intracellular redox state of vascular SMCs. Therefore, it is conceivable that injury-induced ROS generation may increase MMP-9 induction, and perturbations in cell-matrix interactions may result in increased vascular SMC apoptosis. The inhibition of oxidative stress by Tempol treatment will attenuate this cellular pathway. Thus it will be of interest to determine the induction of MMP-9 following injury and the effect of ROS inhibition on this signaling pathway. We postulate that inhibition of MMP-9 induction at the time of injury may prevent early inhibition of medial SMC apoptosis, leading to subsequent decrease in neointimal hyperplasia.

In conclusion, in vivo treatment of diabetic rats with the antioxidant agent Tempol inhibits the induction in apoptosis of medial SMCs and prevents the loss of medial SMC density with its concomitant inhibition of neointima formation. These data suggest that antioxidant therapy with Tempol may prove beneficial to attenuate injury-induced vascular wall remodeling in diabetes.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This work was supported by the American Heart Association and by National Heart, Lung, and Blood Institute Grants HL-14388 and HL-62985 (to F. Miller) and HL-63886, subcontracted through the University of Tennessee.


   ACKNOWLEDGMENTS
 
We thank Dr. Martin Cassell for critically reviewing the manuscript and Paul Reimann for technical assistance with imaging and photography.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Bhalla, Dept. of Anatomy and Cell Biology, Roy J. and Lucille A. Carver College of Medicine, The Univ. of Iowa, Iowa City, IA 52242 (e-mail: ramesh-bhalla{at}uiowa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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  1. Absher PM, Schneider DJ, Baldor LC, Russell JC, and Sobel BE. Increased proliferation of explanted vascular smooth muscle cells: a marker presaging atherogenesis. Atherosclerosis 131: 187–194, 1997.[CrossRef][Web of Science][Medline]
  2. Aoki M, Nata T, Morishita R, Matsushita H, Nakagami H, Yamamoto K, Yamazaki K, Nakabayashi M, Ogihara T, and Kaneda Y. Endothelial apoptosis induced by oxidative stress through activation of NF-{kappa}B: antiapoptotic effect of antioxidant agents on endothelial cells. Hypertension 38: 48–55, 2001.[Abstract/Free Full Text]
  3. Beohar N, Flaherty JD, Davidson CJ, Maynard RC, Robbins JD, Shah AP, Choi JW, MacDonald LA, Jorgensen JP, Pinto JV, Chandra S, Klaus HM, Wang NC, Harris KR, Decker R, and Bonow RO. Antirestenotic effects of a locally delivered caspase inhibitor in a balloon injury model. Circulation 109: 108–113, 2004.[Abstract/Free Full Text]
  4. Best PJ, Hasdai D, Sangiorgi G, Schwartz RS, Holmes DR Jr, Simari RD, and Lerman A. Apoptosis. Basic concepts and implications in coronary artery disease. Arterioscler Thromb Vasc Biol 19: 14–22, 1999.[Abstract/Free Full Text]
  5. Chen SJ, Li H, Durand J, Oparil S, and Chen YF. Estrogen reduces myointimal proliferation after balloon injury of rat carotid artery. Circulation 93: 577–584, 1996.[Abstract/Free Full Text]
  6. Durand E, Mallat Z, Addad F, Vilde F, Desnos M, Guerot C, Tedgui A, and Lafont A. Time courses of apoptosis and cell proliferation and their relationship to arterial remodeling and restenosis after angioplasty in an atherosclerotic rabbit model. J Am Coll Cardiol 39: 1680–1685, 2002.[Abstract/Free Full Text]
  7. Evans JL, Goldfine ID, Maddux BA, and Grodsky GM. Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr Rev 23: 599–622, 2002.[Abstract/Free Full Text]
  8. Fang S, Sharma RV, and Bhalla RC. Enhanced recovery of injury-caused downregulation of paxillin protein by eNOS gene expression in rat carotid artery. Mechanism of NO inhibition of intimal hyperplasia? Arterioscler Thromb Vasc Biol 19: 147–152, 1999.[Abstract/Free Full Text]
  9. Ferns GA, Forster L, Stewart-Lee A, Konneh M, Nourooz-Zadeh J, and Anggard EE. Probucol inhibits neointimal thickening and macrophage accumulation after balloon injury in the cholesterol-fed rabbit. Proc Natl Acad Sci USA 89: 11312–11316, 1992.[Abstract/Free Full Text]
  10. Gelvan D, Saltman P, and Powell SR. Cardiac reperfusion damage prevented by a nitroxide free radical. Proc Natl Acad Sci USA 88: 4680–4684, 1991.[Abstract/Free Full Text]
  11. Griendling KK, Sorescu D, Lassegue B, and Ushio-Fukai M. Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol 20: 2175–2183, 2000.[Abstract/Free Full Text]
  12. Gurjar MV, Deleon J, Sharma RV, and Bhalla RC. Role of reactive oxygen species in IL-1{beta}-stimulated sustained ERK activation and MMP-9 induction. Am J Physiol Heart Circ Physiol 281: H2568–H2574, 2001.[Abstract/Free Full Text]
  13. Han DK, Haudenschild CC, Hong MK, Tinkle BT, Leon MB, and Liau G. Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol 147: 267–277, 1995.[Abstract]
  14. Hwang IS, Ho H, Hoffman BB, and Reaven GM. Fructose-induced insulin resistance and hypertension in rats. Hypertension 10: 512–516, 1987.[Abstract/Free Full Text]
  15. Isner JM, Kearney M, Bortman S, and Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation 91: 2703–2711, 1995.[Abstract/Free Full Text]
  16. Jordan JE, Simandle SA, Tulbert CD, Busija DW, and Miller AW. Fructose-fed rats are protected against ischemia/reperfusion injury. J Pharmacol Exp Ther 307: 1007–1011, 2003.[Abstract/Free Full Text]
  17. Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D, and Reed JC. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA 95: 4997–5002, 1998.[Abstract/Free Full Text]
  18. Karmeli F, Eliakim R, Okon E, Samuni A, and Rachmilewitz D. A stable nitroxide radical effectively decreases mucosal damage in experimental colitis. Gut 37: 386–393, 1995.[Abstract/Free Full Text]
  19. Kluck RM, Esposti MD, Perkins G, Renken C, Kuwana T, Bossy-Wetzel E, Goldberg M, Allen T, Barber MJ, Green DR, and Newmeyer DD. The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol. J Cell Biol 147: 809–822, 1999.[Abstract/Free Full Text]
  20. Kollum M, Kaiser S, Kinscherf R, Metz J, Kubler W, and Hehrlein C. Apoptosis after stent implantation compared with balloon angioplasty in rabbits. Role of macrophages. Arterioscler Thromb Vasc Biol 17: 2383–2388, 1997.[Abstract/Free Full Text]
  21. Kornowski R, Mintz GS, Kent KM, Pichard AD, Satler LF, Bucher TA, Hong MK, Popma JJ, and Leon MB. Increased restenosis in diabetes mellitus after coronary interventions is due to exaggerated intimal hyperplasia. A serial intravascular ultrasound study. Circulation 95: 1366–1369, 1997.[Abstract/Free Full Text]
  22. Kroemer G, Dallaporta B, and Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60: 619–642, 1998.[CrossRef][Web of Science][Medline]
  23. Levine RL, Chen SJ, Durand J, Chen YF, and Oparil S. Medroxyprogesterone attenuates estrogen-mediated inhibition of neointima formation after balloon injury of the rat carotid artery. Circulation 94: 2221–2227, 1996.[Abstract/Free Full Text]
  24. Li PF, Dietz R, and von Harsdorf R. Reactive oxygen species induce apoptosis of vascular smooth muscle cell. FEBS Lett 404: 249–252, 1997.[CrossRef][Web of Science][Medline]
  25. Malik N, Francis SE, Holt CM, Gunn J, Thomas GL, Shepherd L, Chamberlain J, Newman CM, Cumberland DC, and Crossman DC. Apoptosis and cell proliferation after porcine coronary angioplasty. Circulation 98: 1657–1665, 1998.[Abstract/Free Full Text]
  26. Maytin M, Leopold J, and Loscalzo J. Oxidant stress in the vasculature. Curr Atheroscler Rep 1: 156–164, 1999.[Medline]
  27. Miyauchi K, Aikawa M, Tani T, Nakahara K, Kawai S, Nagai R, Okada R, and Yamaguchi H. Effect of probucol on smooth muscle cell proliferation and dedifferentiation after vascular injury in rabbits: possible role of PDGF. Cardiovasc Drugs Ther 12: 251–260, 1998.[CrossRef][Web of Science][Medline]
  28. Oltvai ZN, Milliman CL, and Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74: 609–619, 1993.[CrossRef][Web of Science][Medline]
  29. Pastorino JG, Chen ST, Tafani M, Snyder JW, and Farber JL. The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition. J Biol Chem 273: 7770–7775, 1998.[Abstract/Free Full Text]
  30. Perlman H, Maillard L, Krasinski K, and Walsh K. Evidence for the rapid onset of apoptosis in medial smooth muscle cells after balloon injury. Circulation 95: 981–987, 1997.[Abstract/Free Full Text]
  31. Pollman MJ, Hall JL, and Gibbons GH. Determinants of vascular smooth muscle cell apoptosis after balloon angioplasty injury. Influence of redox state and cell phenotype. Circ Res 84: 113–121, 1999.[Abstract/Free Full Text]
  32. Scarborough NL and Carrier GO. Nifedipine and alpha adrenoceptors in rat aorta. II. Role of extracellular calcium in enhanced alpha-2 adrenoceptor-mediated contraction in diabetes. J Pharmacol Exp Ther 231: 603–609, 1984.[Abstract/Free Full Text]
  33. Schwartz SM, deBlois D, and O’Brien ER. The intima. Soil for atherosclerosis and restenosis. Circ Res 77: 445–465, 1995.[Free Full Text]
  34. Sedlak TW, Oltvai ZN, Yang E, Wang K, Boise LH, Thompson CB, and Korsmeyer SJ. Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proc Natl Acad Sci USA 92: 7834–7838, 1995.[Abstract/Free Full Text]
  35. Shi M, Yang H, Motley ED, and Guo Z. Overexpression of Cu/Zn-superoxide dismutase and/or catalase in mice inhibits aorta smooth muscle cell proliferation. Am J Hypertens 17: 450–456, 2004.[CrossRef][Web of Science][Medline]
  36. Sousa JE, Costa MA, and Sousa AG. What is "the matter" with restenosis in 2002? Circulation 105: 2932–2933, 2002.[Free Full Text]
  37. Spitaler MM and Graier WF. Vascular targets of redox signalling in diabetes mellitus. Diabetologia 45: 476–494, 2002.[CrossRef][Web of Science][Medline]
  38. Tamura K, Kanzaki T, Tashiro J, Yokote K, Mori S, Ueda S, Saito Y, and Morisaki N. Increased atherogenesis in Otsuka Long-Evans Tokushima fatty rats before the onset of diabetes mellitus: association with overexpression of PDGF {beta}-receptors in aortic smooth muscle cells. Atherosclerosis 149: 351–358, 2000.[CrossRef][Web of Science][Medline]
  39. Tribe RM and Poston L. Oxidative stress and lipids in diabetes: a role in endothelium vasodilator dysfunction? Vasc Med 1: 195–206, 1996.[Medline]
  40. Walsh K and Isner JM. Apoptosis in inflammatory-fibroproliferative disorders of the vessel wall. Cardiovasc Res 45: 756–765, 2000.[Abstract/Free Full Text]
  41. Wei GL, Krasinski K, Kearney M, Isner JM, Walsh K, and Andres V. Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty. Circ Res 80: 418–426, 1997.[Web of Science][Medline]
  42. Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB, and Korsmeyer SJ. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292: 727–730, 2001.[Abstract/Free Full Text]
  43. Wolff SP and Dean RT. Glucose autoxidation and protein modification. The potential role of "autoxidative glycosylation" in diabetes. Biochem J 245: 243–250, 1987.[Web of Science][Medline]
  44. Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP, and Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275: 1129–1132, 1997.[Abstract/Free Full Text]



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